Fig. 4

Effects of anlotinib and SAR131675 on the structure and function of tumor blood vessels, tumor stroma and IFP. a–e During the time and dosing window of anlotinib used in this experiment, tumor vessel normalization did not occur. Immunohistochemistry staining for endothelial cells (CD31, brown) (Scale bar, 100 μm), immunofluorescence staining for endothelial cells (CD31, red) and pericytes (NG2, green) (Scale bar, 80 μm), fluorescence images of Dylight^@488-lectin-perfused (green) tumor blood vessels (CD31, red) (Scale bar, 40 μm), and representative images of HIF-1α (brown) immunohistochemical staining (Scale bar, 40 μm) in 4T1 tumor sections from mice treated with saline or anlotinib are shown in (a). Quantitative analysis of tumor vascular density (b), pericyte coverage (c; NG2⁺CD31⁺ area percentage of the total CD31⁺ area), perfused vessels (d; lectin⁺ CD31⁺ area percentage of the total CD31⁺ area), and HIF-1α area (e) as shown in (a) (n = 9 or n = 12; images were from three mice per group). f–h SAR131675 did not influence the density and function of tumor blood vessels. Immunohistochemistry staining for endothelial cells (CD31, brown) and HIF-1α (brown) in 4T1 tumor sections from mice treated with saline or SAR131675 are shown in (f). Scale bar, 100 μm in the upper panels and 20 μm in the lower panels. Quantification of tumor vascular density (g) and HIF-1α area (h) as shown in (f) (n = 9; images were from three mice per group). i–k Anlotinib and SAR131675 did not modulate tumor stroma. Histological studies with trichrome staining of collagen and immunohistochemical staining of fibronectin in tumor (i). Scale bar, 50 μm. Quantitative analysis of collagen (j) and fibronectin (k) (n = 9; images were from three mice per group). l Tumor IFP of tumor-bearing mice treated with saline, anlotinib, or SAR131675 for 10 consecutive days (n = 9). The data are shown as the mean ± s.d. ns no significance, ***p < 0.001